Assay method using magnetic particles

Abstract

Method for assaying a target analyte in a biological sample in liquid medium, comprising: contacting the sample with first magnetic particles bearing a first receptor specific to a first analyte site of attachment to form first complexes; applying a first magnetic field to locally combine the formed complexes formed and optionally to agglomerate interfering complexes to form interfering aggregates; negating the applied magnetic field; adding second magnetic particles to a liquid medium that bear a second receptor specific to a second analyte site of attachment; measuring a first quantity of interfering aggregates; applying a second magnetic field to form second complexes; measuring a second quantity of the collective amount of interfering aggregates and second complexes to determine an amount of formed second complexes as a function of the first quantity, and deducing the amount of analyte present in the sample and, optionally, the amount of interfering analyte.

1 . A method for assaying a target analyte in a biological sample in a liquid medium, comprising the following steps:
 a. contacting the biological sample with first magnetic particles bearing a first receptor specific to a first site of attachment of the target analyte so as to form first complexes by bonding of the first magnetic particles with the target analyte this contacting being accompanied, when an interfering analyte is present in the sample, with the formation of interfering complexes by the non-specific bonding of said interfering analyte to the first magnetic particles;   b. applying a first magnetic field, and maintaining it, so as to locally combine all of the complexes formed in step a., and, if applicable, agglomerate interfering complexes with one another to form interfering aggregates;   c. negating the first magnetic field applied in step b. and adding in the liquid medium second magnetic particles bearing a second receptor specific to a second site of attachment of the analyte target;   d. measuring a first quantity representative of the amount of interfering aggregates in the liquid medium, to identify the presence or absence of said interfering aggregates;   e. applying a second magnetic field so as to form second complexes by bonding the first complexes with second magnetic particles; and   f. measuring a second quantity representative of the collective amount of interfering aggregates and of second complexes in the liquid medium so as to determine the amount of second complexes formed in step e. as a function of the first quantity for deducing therefrom the amount of target analyte present in the biological sample and, if applicable, the amount of interfering analyte.   
     
     
         2 . The assay method according to  claim 1 , wherein step f. comprises calculating the difference between the second quantity measured at this step f. and the first quantity measured in step d. 
     
     
         3 . The method according to  claim 1 , wherein steps c. and d. are performed substantially simultaneously. 
     
     
         4 . The method according to  claim 1 , wherein step d. is carried out after negation of the first magnetic field, before adding second magnetic particles in the liquid medium. 
     
     
         5 . The method according to  claim 1 , wherein holding the magnetic field applied in step b. lasts less than 5 minutes, preferably less than 3 minutes. 
     
     
         6 . The method according to  claim 1 , wherein applying the second magnetic field includes a magnetisation at 8 mT for 1 second, followed by three successive sequences of magnetisations and cuts as follows: 15 mT for 60 seconds, 0 mT for 28 seconds, 8 mT for 1 second, 0 mT for 1 second. 
     
     
         7 . The method according to  claim 1 , wherein measuring the first and/or second magnitude is selected from the group consisting of measurement by turbidimetry, measurement by nephelometry and measurement by counting. 
     
     
         8 . The method according to  claim 1 , wherein step a. includes adding a diluent so as to dilute the biological sample, the ratio between the sample and the diluent being greater than or equal to 1:10 (volume). 
     
     
         9 . The method according to  claim 1 , wherein step b. includes extracting the first complexes from the liquid medium. 
     
     
         10 . The method according to  claim 1 , wherein step b. includes a sub-step b1. comprising removing the liquid medium followed by adding a reaction buffer, and preferably at least one washing between the removal of the liquid medium and the addition of the reaction buffer. 
     
     
         11 . The method according to  claim 10 , wherein the reaction buffer comprises at least one anti-aggregation agent and/or a cell lysing agent. 
     
     
         12 . The method according to  claim 1 , wherein the sample is a whole blood sample. 
     
     
         13 . The method according to  claim 1 , wherein the interfering aggregates are identified when the first quantity exceeds a predefined threshold quantity. 
     
     
         14 . A device arranged to carry out the method according to  claim 1 .